1. Field of the Invention
This invention relates generally to gonadotropin-releasing hormone (GnRH) receptor antagonists, and to methods of treating disorders by administration of such antagonists to a warm-blooded animal in need thereof.
2. Description of the Related Art
Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH), is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) that plays an important role in human reproduction. GnRH is released from the hypothalamus and acts on the pituitary gland to stimulate the biosynthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH released from the pituitary gland is responsible for the regulation of gonadal steroid production in both males and females, while FSH regulates spermatogenesis in males and follicular development in females.
Due to its biological importance, synthetic antagonists and agonists to GnRH have been the focus of considerable attention, particularly in the context of prostate cancer, breast cancer, endometriosis, uterine leiomyoma, and precocious puberty. For example, peptidic GnRH agonists, such as leuprorelin (pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), have been used to treat such conditions. Such agonists appear to function by binding to the GnRH receptor in the pituitary gonadotropins, thereby inducing the synthesis and release of gonadotropins. Chronic administration of GnRH agonists depletes gonadotropins and subsequently down-regulates the receptor, resulting in suppression of steroidal hormones after some period of time (e.g., on the order of 2-3 weeks following initiation of chronic administration).
In contrast, GnRH antagonists are believed to suppress gonadotropins from the onset, and thus have received the most attention over the past two decades. To date, some of the primary obstacles to the clinical use of such antagonists have been their relatively low bioavailability and adverse side effects caused by histamine release. However, several peptidic antagonists with low histamine release properties have been reported, although they still must be delivered via sustained delivery routes (such as subcutaneous injection or intranasal spray) due to limited bioavailability.
In view of the limitations associated with peptidic GnRH antagonists, a number of nonpeptidic compounds have been proposed. For example, Cho et al. (J. Med. Chem. 41:4190-4195, 1998) discloses thieno[2,3-b]pyridin-4-ones for use as GnRH receptor antagonists; U.S. Pat. Nos. 5,780,437 and 5,849,764 teach substituted indoles as GnRH receptor antagonists (as do published PCTs WO 97/21704, 98/55479, 98/55470, 98/55116, 98/55119, 97/21707, 97/21703 and 97/21435); published PCT WO 96/38438 discloses tricyclic diazepines as GnRH receptor antagonists; published PCTs WO97/14682, 97/14697 and 99/09033 disclose quinoline and thienopyridine derivatives as GnRH antagonists; published PCTs WO 97/44037, 97/44041, 97/44321 and 97/44339 teach substituted quinolin-2-ones as GnRH receptor antagonists; and published PCT WO 99/33831 discloses certain phenyl-substituted fused nitrogen-containing bicyclic compounds as GnRH receptor antagonists.
While significant strides have been made in this field, there remains a need in the art for effective small molecule GnRH receptor antagonists. There is also a need for pharmaceutical compositions containing such GnRH receptor antagonists, as well as methods relating to the use thereof to treat, for example, sex-hormone related conditions. The present invention fulfills these needs, and provides other related advantages.
In brief, this invention is generally directed to gonadotropin-releasing hormone (GnRH) receptor antagonists, as well as to methods for their preparation and use, and to pharmaceutical compositions containing the same. More specifically, the GnRH receptor antagonists of this invention are compounds having the following general structure (I): 
including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein A, R1, R2, R3a, R3b, R4, R5, R6, and n are as defined below.
The GnRH receptor antagonists of this invention have utility over a wide range of therapeutic applications, and may be used to treat a variety of sex-hormone related conditions in both men and women, as well as a mammal in general (also referred to herein as a xe2x80x9csubjectxe2x80x9d). For example, such conditions include endometriosis, uterine fibroids, polycystic ovarian disease, hirsutism, precocious puberty, gonadal steroid-dependent neoplasia such as cancers of the prostate, breast and ovary, gonadotrophe pituitary adenomas, sleep apnea, irritable bowel syndrome, premenstrual syndrome, benign prostatic hypertrophy, contraception and infertility (e.g., assisted reproductive therapy such as in vitro fertilization). The compounds of this invention are also useful as an adjunct to treatment of growth hormone deficiency and short stature, and for the treatment of systemic lupus erythematosis. The compounds are also useful in combination with androgens, estrogens, progesterones, and antiestrogens and antiprogestogens for the treatment of endometriosis, fibroids, and in contraception, as well as in combination with an angiotensin-converting enzyme inhibitor, an angiotensin II-receptor antagonist, or a renin inhibitor for the treatment of uterine fibroids. In addition, the compounds may be used in combination with bisphosphonates and other agents for the treatment and/or prevention of disturbances of calcium, phosphate and bone metabolism, and in combination with estrogens, progesterones and/or androgens for the prevention or treatment of bone loss or hypogonadal symptoms such as hot flashes during therapy with a GnRH antagonist.
The methods of this invention include administering an effective amount of a GnRH receptor antagonist, preferably in the form of a pharmaceutical composition, to a mammal in need thereof. Thus, in still a further embodiment, pharmaceutical compositions are disclosed containing one or more GnRH receptor antagonists of this invention in combination with a pharmaceutically acceptable carrier and/or diluent.
These and other aspects of the invention will be apparent upon reference to the following detailed description. To this end, various references are set forth herein which describe in more detail certain background information, procedures, compounds and/or compositions, and are each hereby incorporated by reference in their entirety.
As mentioned above, the present invention is directed generally to compounds useful as gonadotropin-releasing hormone (GnRH) receptor antagonists. The compounds of this invention have the following structure (I): 
including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein:
A is N or CR7;
n is 2, 3 or 4;
R1 and R2 are the same or different and independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, xe2x80x94C(R8)(xe2x95x90NR9) or xe2x80x94C(NR10R11)(xe2x95x90NR9);
or R1 and R2 taken together with the nitrogen atom to which they are attached form a heterocycle or a substituted heterocycle;
R3a and R3b are the same or different and, at each occurrence, independently hydrogen, alkyl, substituted alkyl, alkoxy, alkylthio, alkylamino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, xe2x80x94COOR12 or xe2x80x94CONR10R11;
or R3a and R3b taken together with the carbon atom to which they are attached form a homocycle, substituted homocycle, heterocycle or substituted heterocycle;
or R3a and the carbon to which it is attached taken together with R1 and the nitrogen to which it is attached form a heterocycle or substituted heterocycle;
R4 is arylalkyl, substituted arylalkyl, heteroarylalkyl or substituted heteroarylalkyl;
R5 is hydrogen, alkyl, or substituted alkyl;
R6 is aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
R7 is hydrogen, alkyl, or substituted alkyl;
R8 is independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl;
R9 is independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl;
R10 and R11 are the same or different independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl; and
R12 is hydrogen, alkyl, or substituted alkyl.
As used herein, the above terms have the following meaning:
xe2x80x9cAlkylxe2x80x9d means a straight chain or branched, noncyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon containing from 1 to 10 carbon atoms, while the term xe2x80x9clower alkylxe2x80x9d has the same meaning as alkyl but contains from 1 to 6 carbon atoms. The term xe2x80x9chigher alkylxe2x80x9d has the same meaning as alkyl but contains from 2 to 10 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like. Cyclic alkyls are also referred to herein as a xe2x80x9chomocyclesxe2x80x9d or xe2x80x9chomocyclic rings.xe2x80x9d Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an xe2x80x9calkenylxe2x80x9d or xe2x80x9calkynylxe2x80x9d, respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.
xe2x80x9cArylxe2x80x9d means an aromatic carbocyclic moiety such as phenyl or naphthyl.
xe2x80x9cArylalkylxe2x80x9d means an alkyl having at least one alkyl hydrogen atoms replaced with an aryl moiety, such as benzyl, xe2x80x94(CH2)2phenyl, xe2x80x94(CH2)3phenyl, xe2x80x94CH(phenyl)2, and the like.
xe2x80x9cHeteroarylxe2x80x9d means an aromatic heterocycle ring of 5- to 10 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and bicyclic ring systems. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.
xe2x80x9cHeteroarylalkylxe2x80x9d means an alkyl having at least one alkyl hydrogen atom replaced with a heteroaryl moiety, such as xe2x80x94CH2pyridinyl, xe2x80x94CH2pyrimidinyl, and the like.
xe2x80x9cHeterocyclexe2x80x9d (also referred to herein as a xe2x80x9cheterocyclic ringxe2x80x9d) means a 4- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined above. Thus, in addition to the heteroaryls listed above, heterocycles also include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
xe2x80x9cHeterocyclealkylxe2x80x9d means an alkyl having at least one alkyl hydrogen atom replaced with a heterocycle, such as xe2x80x94CH2morpholinyl, and the like.
xe2x80x9cHomocyclexe2x80x9d (also referred to herein as xe2x80x9chomocyclic ringxe2x80x9d) means a saturated or unsaturated (but not aromatic) carbocyclic ring containing from 3-7 carbon atoms, such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene, and the like.
The term xe2x80x9csubstitutedxe2x80x9d as used herein means any of the above groups (i.e., alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, homocycle, heterocycle and/or heterocyclealkyl) wherein at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (xe2x80x9cxe2x95x90Oxe2x80x9d) two hydrogen atoms are replaced. When substituted one or more of the above groups are substituted, xe2x80x9csubstituentsxe2x80x9d within the context of this invention include halogen, hydroxy, oxo, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle and heterocyclealkyl, as well as xe2x80x94NRaRb, xe2x80x94NRaC(xe2x95x90O)Rb, xe2x80x94NRaC(xe2x95x90O)NRaNRb, xe2x80x94NRaC(xe2x95x90O)ORb xe2x80x94NRaSO2Rb, C(xe2x95x90O)Ra, xe2x80x94C(xe2x95x90O)ORa, xe2x80x94C(xe2x95x90O)NRaRb, xe2x80x94ORa, xe2x80x94SRa, xe2x80x94SORa, xe2x80x94S(xe2x95x90O)2Ra, xe2x80x94OS(xe2x95x90O)2Ra and xe2x80x94S(xe2x95x90O)2ORa. In addition, the above substituents may be further substituted with one or more of the above substituents, such that the substituent substituted alky, substituted aryl, substituted arylalkyl, substituted heterocycle or substituted heterocyclealkyl. Ra and Rb in this context may be the same or different and independently hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl.
xe2x80x9cHalogenxe2x80x9d means fluoro, chloro, bromo and iodo.
xe2x80x9cHaloalkylxe2x80x9d means an alkyl having at least one hydrogen atom replaced with halogen, such as trifluoromethyl and the like.
xe2x80x9cAlkoxyxe2x80x9d means an alkyl moiety attached through an oxygen bridge (i.e., xe2x80x94O-alkyl) such as methoxy, ethoxy, and the like.
xe2x80x9cAlkylthioxe2x80x9d means an alkyl moiety attached through a sulfur bridge (i.e., xe2x80x94Sxe2x80x94 alkyl) such as methylthio, ethylthio, and the like.
xe2x80x9cAlkylsulfonylxe2x80x9d means an alkyl moiety attached through a sulfonyl bridge (i.e., xe2x80x94SO2-alkyl) such as methylsulfonyl, ethylsulfonyl, and the like.
xe2x80x9cAlkylaminoxe2x80x9d and xe2x80x9cdialkylaminoxe2x80x9d mean one or two alkyl moiety attached through a nitrogen bridge (i.e., xe2x80x94N-alkyl) such as methylamino, ethylamino, dimethylamino, diethylamino, and the like.
With regard to the xe2x80x9cR1R2N(CR3aR3b)nxe2x80x94xe2x80x9d moiety of structure (I), n may be 2, 3 or 4. Accordingly, this moiety may be represented by the following structure (i) when n is 2, (ii) when n is 3, and structure (iii) when n is 4: 
wherein each occurrence of R3a and R3b above may be the same or different, and are as defined above. For example, when each occurrence of R3a and R3b in structures (i), (ii) and (iii) is hydrogen, the xe2x80x9cR1R2N(CR3aR3b)nxe2x80x94xe2x80x9d moiety has the structure R1R2N(CH2)2xe2x80x94, R1R2N(CH2)3xe2x80x94 and R1R2N(CH2)4xe2x80x94, respectively.
The compounds of the present invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. However in general, the compounds of structure (I) above may be made by the following Reaction Schemes. All substituents in the following Reaction Schemes are as defined above unless indicated otherwise. 
The compounds of structure (I) may generally be referred to as substituted 2H-pyridazin-3-one and 1H-pyridin-2-one compounds, representative compounds of which include the following:
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-difluoro-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-dichloro-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-chloro-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-trifluoromethyl-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-methylsulfonyl-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-chloro-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-trifluoromethyl-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-methylsulfonyl-benzyl)-4-(2-fluoro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-difluoro-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-dichloro-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-chloro-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-trifluoromethyl-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-methylsulfonyl-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-chloro-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-trifluoromethyl-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-methylsulfonyl-benzyl)-4-(2-fluoro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-difluoro-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-dichloro-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-chloro-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-trifluoromethyl-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-methylsulfonyl-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-chloro-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-trifluoromethyl-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-methylsulfonyl-benzyl)-4-(2-chloro-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-difluoro-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-dichloro-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-chloro-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-trifluoromethyl-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-methylsulfonyl-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-chloro-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-trifluoromethyl-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-methylsulfonyl-benzyl)-4-(3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-difluoro-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2,6-dichloro-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-chloro-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-trifluoromethyl-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-6-methylsulfonyl-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-fluoro-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-chloro-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-trifluoromethyl-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
2-(2-Amino-2-phenyl-ethyl)-6-(2-methylsulfonyl-benzyl)-4-(2-chloro-3-methoxy-phenyl)-5-methyl-2H-pyridazin-3-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-difluoro-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-dichloro-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-chloro-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-trifluoromethyl-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-methylsulfonyl-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-chloro-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-trifluoromethyl-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-methyl sulfonyl-benzyl)-3-(2-fluoro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-difluoro-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-dichloro-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-chloro-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-trifluoromethyl-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-methylsulfonyl-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-chloro-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-trifluoromethyl-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-methylsulfonyl-benzyl)-3-(2-fluoro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-difluoro-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-dichloro-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-chloro-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-trifluoromethyl-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-methylsulfonyl-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-chloro-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-trifluoromethyl-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-methylsulfonyl-benzyl)-3-(2-chloro-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-difluoro-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-dichloro-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-chloro-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-trifluoromethyl-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-methylsulfonyl-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-chloro-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-trifluoromethyl-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-methyl sulfonyl-benzyl)-3-(3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-difluoro-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2,6-dichloro-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-chloro-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-trifluoromethyl-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-6-methylsulfonyl-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-fluoro-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-chloro-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one;
1-(2-Amino-2-phenyl-ethyl)-5-(2-trifluoromethyl-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one; and
1-(2-Amino-2-phenyl-ethyl)-5-(2-methylsulfonyl-benzyl)-3-(2-chloro-3-methoxy-phenyl)-4,6-dimethyl-1H-pyridin-2-one.
In addition, representative compounds of the present invention also include those compounds where the primary amine of the above named compounds is substituted with a substituted alkyl group or a cycloalkyl group. As described in the examples, one method of alkylating amines and amides is by reductive alkylation. There are many alternative methods well known in the chemical arts for accomplishing the reductive alkylation procedure, and there are many alternative alkylation methods. When an aldehyde, ketone, carboxylic acid, or acid chloride is treated with a primary or secondary amine in the presence of a reducing agent reductive alkylation may take place. Suitable reducing agents include (but are not limited to) sodium borohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen gas and a hydrogenation catalyst, zinc and hydrochloric acid, iron pentacarbonyl and alcoholic potassium hydroxide, formic acid, pyridine borohydride. Amines and amides may also be alkylated by the reaction of formaldehyde and a Mannich base or by the nucleophilic displacement of an alkyl halide or other leaving groups. As an example, the Mitsunobu reaction allows the alkylation of amines with primary or secondary alcohols and carboxylic acids by activation of the hydroxyl group with triphenylphosphine to form the leaving group triphenylphoshine oxide. Other commonly used alkylation methods are described in March, Advanced Organic Chemistry, 4th Ed., pp 1276-1277 (1992).
The compounds of the present invention may generally be utilized as the free acid or free base. Alternatively, the compounds of this invention may be used in the form of acid or base addition salts. Acid addition salts of the free amino compounds of the present invention may be prepared by methods well known in the art, and may be formed from organic and inorganic acids. Suitable organic acids include maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Base addition salts included those salts that form with the carboxylate anion and include salts formed with organic and inorganic cations such as those chosen from the alkali and alkaline earth metals (for example, lithium, sodium, potassium, magnesium, barium and calcium), as well as the ammonium ion and substituted derivatives thereof (for example, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, and the like). Thus, the term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d of structure (I) is intended to encompass any and all acceptable salt forms.
In addition, prodrugs are also included within the context of this invention. Prodrugs are any covalently bonded carriers that release a compound of structure (I) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound. Prodrugs include, for example, compounds of this invention wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to a patient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus, representative examples of prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol and amine functional groups of the compounds of structure (I). Further, in the case of a carboxylic acid (xe2x80x94COOH), esters may be employed, such as methyl esters, ethyl esters, and the like.
With regard to stereoisomers, the compounds of structure (I) may have chiral centers and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers. Compounds of structure (I) may also possess axial chirality, which may result in atropisomers. All such isomeric forms are included within the present invention, including mixtures thereof. Furthermore, some of the crystalline forms of the compounds of structure (I) may exist as polymorphs, which are included in the present invention. In addition, some of the compounds of structure (I) may also form solvates with water or other organic solvents. Such solvates are similarly included within the scope of this invention.
The effectiveness of a compound as a GnRH receptor antagonist may be determined by various assay methods. Suitable GnRH antagonists of this invention are capable of inhibiting the specific binding of GnRH to its receptor and antagonizing activities associated with GnRH. For example, inhibition of GnRH stimulated LH release in immature rats may be measured according to the method of Vilchez-Martinez (Endocrinology 96:1130-1134, 1975). Briefly, twenty-five day old male Spraque-Dawley rats are administered an GnRH antagonist in saline or other suitable formulation by oral gavage, sub-cutaneous injection, or intravenous injection. This is followed by sub-cutaneous injection of 200 ng GnRH in 0.2 ml saline. Thirty minutes after the last injection, the animals are decapitated and trunk blood collected. After centrifugation, the separated plasma is stored at xe2x88x9220xc2x0 C. until determination of the LH and FSH by radioimmunoassay. Other techniques for determining the activity of GnRH receptor antagonists are well known in the field, such as the use of cultured pituitary cells for measuring GnRH activity (Vale et al., Endocrinology 91:562-572, 1972), and a technique for measuring radioligand binding to rat pituitary membranes (Perrin et al., Mol. Pharmacol. 23:44-51, 1983).
For example, effectiveness of a compound as a GnRH receptor antagonist may be determined by one or more of the following assays.
Rat Anterior Pituitary Cell Culture Assay of GnRH Antagonists
Anterior pituitary glands are collected from 7-week-old female Sprague-Dawley rats and the harvested glands digested with collagenase in a dispersion flask for 1.5 hr at 37xc2x0 C. After collagenase digestion, the glands are further digested with neuraminidase for 9 min at 37xc2x0 C. The digested tissue is then washed with 0.1% BSA/McCoy""s 5A medium, and the washed cells suspended in 3% FBS/0.1 BSA/McCoy""s 5A medium and plated into 96-well tissue culture plates at a cell density of 40,000 cells per well in 200 xcexcl medium. The cells are then incubated at 37xc2x0 C. for 3 days. One pituitary gland normally yields one 96-well plate of cells, which can be used for assaying three compounds. For assay of an GnRH antagonist, the incubated cells are first washed with 0.1% BSA/McCoy""s 5A medium once, followed by addition of the test sample plus 1 nM GnRH in 200 xcexcl 0.1% BSA/McCoy""s 5A medium in triplicate wells. Each sample is assayed at 5-dose levels to generate a dose-response curve for determination of its potency on the inhibition of GnRH stimulated LH and/or FSH release. After 4-hr incubation at 37xc2x0 C., the medium is harvested and the level of LH and/or FSH secreted into the medium determined by RIA.
RIA of LH and FSH
For determination of the LH levels, each sample medium is assayed in duplicates and all dilutions are done with RIA buffer (0.01M sodium phosphate buffer/0.15M NaCl/1% BSA/0.01% NaN3, pH 7.5) and the assay kit is obtained from the Nation Hormone and Pituitary Program supported by NIDDK. To a 12xc3x9775 mm polyethylene test tube is added 100 xcexcl of sample medium diluted 1:5 or rLH standard in RIA buffer and 100 xcexcl of [1251]-labeled rLH (xcx9c30,000 cpm) plus 100 xcexcl of rabbit anti-rLH antibody diluted 1:187,500 and 100 xcexcl RIA buffer. The mixture is incubated at room temperature over-night. In the next day, 100 xcexcl of goat anti-rabbit IgG diluted 1:20 and 100 xcexcl of normal rabbit serum diluted 1:1000 are added and the mixture incubated for another 3 hr at room temperature. The incubated tubes are then centrifuged at 3,000 rpm for 30 min and the supernatant removed by suction. The remaining pellet in the tubes is counted in a gamma-counter. RIA of FSH is done in a similar fashion as the assay for LH with substitution of the LH antibody by the FSH antibody diluted 1:30,000 and the labeled rLH by the labeled rFSH.
Radio-iodination of GnRH Peptide
The GnRH analog is labeled by the chloramine-T method. To 10 xcexcg of peptide in 20 xcexcl of 0.5M sodium phosphate buffer, pH 7.6, is added 1 mCi of Na125I, followed by 22.5 xcexcg chloramine-T and the mixture vortexed for 20 sec. The reaction is stopped by the addition of 60 xcexcg sodium metabisulfite and the free iodine is removed by passing the iodinated mixture through a C-8 Sep-Pak cartridge (Millipore Corp., Milford, Mass.). The peptide is eluted with a small volume of 80% acetonitrile/water. The recovered labeled peptide is further purified by reverse phase HPLC on a Vydac C-18 analytical column (The Separations Group, Hesperia, Calif.) on a Beckman 334 gradient HPLC system using a gradient of acetonitrile in 0.1% TFA. The purified radioactive peptide is stored in 0.1% BSA/20% acetonitrile/0.1% TFA at xe2x88x9280xc2x0 C. and can be used for up to 4 weeks.
GnRH Receptor Membrane Binding Assay
Cells stably, or transiently, transfected with GnRH receptor expression vectors are harvested, resuspended in 5% sucrose and homogenized using a polytron homogenizer (2xc3x9715 sec). Nucleii are removed by centrifugation (3000xc3x97 g for 5 min.), and the supernatant centrifuged (20,000xc3x97 g for 30 min, 4xc2x0 C.) to collect the membrane fraction. The final membrane preparation is resuspended in binding buffer (10 mM Hepes (pH 7.5), 150 mM NaCl, and 0.1% BSA) and stored at xe2x88x9270xc2x0 C. Binding reactions are performed in a Millipore MultiScreen 96-well filtration plate assembly with polyethylenimine coated GF/C membranes. The reaction is initiated by adding membranes (40 ug protein in 130 ul binding buffer) to 50 ul of [125I]-labeled GnRH peptide (xcx9c100,000 cpm), and 20 ul of competitor at varying concentrations. The reaction is terminated after 90 minutes by application of vacuum and washing (2xc3x97) with phosphate buffered saline. Bound radioactivity is measured using 96-well scintillation counting (Packard Topcount) or by removing the filters from the plate and direct gamma counting. Ki values are calculated from competition binding data using non-linear least squares regression using the Prism software package (GraphPad Software).
Activity of GnRH receptor antagonists are typically calculated from the IC50 as the concentration of a compound necessary to displace 50% of the radiolabeled ligand from the GnRH receptor, and is reported as a xe2x80x9cKixe2x80x9d value calculated by the following equation:       K    i    =            I      ⁢              xe2x80x83            ⁢              C        50                    1      +              L        /                  K          D                    
where L=radioligand and KD=affinity of radioligand for receptor (Cheng and Prusoff, Biochem. Pharmacol. 22:3099, 1973). GnRH receptor antagonists of this invention have a Ki of 100 xcexcM or less. In a preferred embodiment of this invention, the GnRH receptor antagonists have a Ki of less than 10 xcexcM, and more preferably less than 1 xcexcM, and even more preferably less than 0.1 xcexcM (i.e., 100 nM).
As mentioned above, the GnRH receptor antagonists of this invention have utility over a wide range of therapeutic applications, and may be used to treat a variety of sex-hormone related conditions in both men and women, as well as mammals in general. For example, such conditions include endometriosis, uterine fibroids, polycystic ovarian disease, hirsutism, precocious puberty, gonadal steroid-dependent neoplasia such as cancers of the prostate, breast and ovary, gonadotrophe pituitary adenomas, sleep apnea, irritable bowel syndrome, premenstrual syndrome, benign prostatic hypertrophy, contraception and infertility (e.g., assisted reproductive therapy such as in vitro fertilization).
The compounds of this invention are also useful as an adjunct to treatment of growth hormone deficiency and short stature, and for the treatment of systemic lupus erythematosis.
In addition, the compounds are useful in combination with androgens, estrogens, progesterones, and antiestrogens and antiprogestogens for the treatment of endometriosis, fibroids, and in contraception, as well as in combination with an angiotensin-converting enzyme inhibitor, an angiotensin II-receptor antagonist, or a renin inhibitor for the treatment of uterine fibroids. The compounds may also be used in combination with bisphosphonates and other agents for the treatment and/or prevention of disturbances of calcium, phosphate and bone metabolism, and in combination with estrogens, progesterones and/or androgens for the prevention or treatment of bone loss or hypogonadal symptoms such as hot flashes during therapy with a GnRH antagonist.
In another embodiment of the invention, pharmaceutical compositions containing one or more GnRH receptor antagonists are disclosed. For the purposes of administration, the compounds of the present invention may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present invention comprise a GnRH receptor antagonist of the present invention and a pharmaceutically acceptable carrier and/or diluent. The GnRH receptor antagonist is present in the composition in an amount that is effective to treat a particular disorderxe2x80x94that is, in an amount sufficient to achieve GnRH receptor antagonist activity, and preferably with acceptable toxicity to the patient. Typically, the pharmaceutical compositions of the present invention may include a GnRH receptor antagonist in an amount from 0.1 mg to 250 mg per dosage depending upon the route of administration, and more typically from 1 mg to 60 mg. Appropriate concentrations and dosages can be readily determined by one skilled in the art.
Pharmaceutically acceptable carrier and/or diluents are familiar to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers and/or diluents include saline and sterile water, and may optionally include antioxidants, buffers, bacteriostats and other common additives. The compositions can also be formulated as pills, capsules, granules, or tablets that contain, in addition to a GnRH receptor antagonist, diluents, dispersing and surface active agents, binders, and lubricants. One skilled in this art may further formulate the GnRH receptor antagonist in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington""s Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.
In another embodiment, the present invention provides a method for treating sex-hormone related conditions as discussed above. Such methods include administering of a compound of the present invention to a warm-blooded animal in an amount sufficient to treat the condition. In this context, xe2x80x9ctreatxe2x80x9d includes prophylactic administration. Such methods include systemic administration of a GnRH receptor antagonist of this invention, preferably in the form of a pharmaceutical composition as discussed above. As used herein, systemic administration includes oral and parenteral methods of administration. For oral administration, suitable pharmaceutical compositions of GnRH receptor antagonists include powders, granules, pills, tablets, and capsules as well as liquids, syrups, suspensions, and emulsions. These compositions may also include flavorants, preservatives, suspending, thickening and emulsifying agents, and other pharmaceutically acceptable additives. For parental administration, the compounds of the present invention can be prepared in aqueous injection solutions which may contain, in addition to the GnRH receptor antagonist, buffers, antioxidants, bacteriostats, and other additives commonly employed in such solutions.
The following example is provided for purposes of illustration, not limitation. In summary, the GnRH receptor antagonists of this invention may be assayed by the general methods disclosed above, while the following Examples disclose the synthesis of representative compounds of this invention.